The proposal has two foci: the mechanism of cell division and the mechanisms by which proteins assemble into membranes. Cell division will be investigated by examining three low abundance bitopic cytoplasmic membrane proteins essential for septum formation and division, FtsI, FtsL, and FtsQ. The fts genes (for temperature sensitive filamentation) are genes in which mutations can block cell division at a non-permissive temperature). The PI has used immunofluorescence microscopy (IFM) to show that a one of these proteins (FtsI) is localized to the septum and proposes similar studies on FtsL and FtsQ. These three proteins somehow cooperate to trigger assembly of a cytoplasmic ring of the soluble protein FtsZ. The FtsZ ring eventually contracts and appears to drive cell division. FtsI, FtsL, and FtsQ are anchored in the membrane by a single membrane spanning segment (MSS), contain short cytoplasmic N-terminal regions (23-37 aa) and more substantial periplasmic segments. FtsL's pepiplasmic domain appears to be a leucine zipper an FtsI's, and penicillin-binding domain that is involved in peptidoglycan synthesis at the septum. By swapping domains among these three proteins and substituting in the MSS from MalF, the PI has determined that only FtsQ can function with heterologous Mss or cytoplasmic domains. The PI proposes to determine whether the location of each protein is dependent on the others (by turning off their expression in specially constructed strains) and on FtsZ. By comparing proteins with various segments replaced and by mutagenesis, he will determine which regions of each are required for their own and for each others localization. He proposes direct tests of the possible role of the leucine zipper in forming homodimers of FtsL or heterodimers that may be involved in localization or function. With these tools in hand, the PI proposes to use second-site suppressor analysis to identify protein-protein interactions essential for cell division. In the second part of the proposal, the PI will use genetic screens and selections to identify genes required for the assembly of proteins into the cytoplasmic membrane. These genes may code for novel cellular components required for this process including those that recognize basic amino acids as cytoplasmic anchors.